Chloramphenicol acetyl transferase (cat)-defective somatostatin fusion protein and uses thereof

ABSTRACT

Chimeric somatostatin-based polypeptides, polynucleotides used to encode the polypeptides, the methods for isolating and producing the polypeptides and the uses thereof are provided. In addition, low cost adjuvants for enhanced immunogenic response are provided. Vaccinations that include both chimeric somatostatin-based polypeptides and novel adjuvants are included, useful in facilitating farm animal productivity.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 111(a) ofPCT/US2008/068195, filed on Jun. 25, 2008, entitled “ChloramphenicolAcetyl Transferase (CAT)-Defective Somatostatin Fusion Protein And UsesThereof” which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to chimeric somatostatin-basedpolypeptides, polynucleotides used to encode the polypeptides, the meansfor isolating and producing the polypeptides, and to the uses thereof.The present invention also relates to novel adjuvants and immunizationcompositions for enhanced immunogenicity of, for example, chimericsomatostatin-based polypeptides of the invention as well as to otherlike antigens.

BACKGROUND OF THE INVENTION

Somatostatin (also known as growth hormone inhibiting hormone or GHIH)is a peptide hormone produced in the hypothalamus as well as certainportions of the digestive system. Somatostatin is generally involved inregulation of the endocrine system via interactions withG-protein-coupled somatostatin receptors. This somatostatin-basedsignaling cascade leads to a number of actions spread throughout thebody.

Relevant to aspects of the present invention, somatostatin is known toinhibit the release of growth hormone and thyroid stimulating hormonefrom the anterior pituitary. (Patel Y C and Srikant C B, Somatostatinand its receptors Adv Mol Cell Endocrinol, 1999. 3 43-73). Otherhormones inhibited by somatostatin include insulin, glucagon, secretin,gastrin, pepsin, maletin, etc. (Patel Y C and Srikant C B Somatostatinand its receptors Adv Mol Cell Endocrinol, 1999. 3 43-73). The abilityof somatostatin to regulate so many factors/hormones necessary forgrowth and utilization of food has made somatostatin a central targetfor controlling animal growth in the animal husbandry field, i.e.,inhibiting somatostatin results in increased levels of growth hormonebeing present in a target animal and thereby results in animals withenhanced capacity to produce milk, to provide greater amounts of meat,etc.

In particular, immunization of animals to somatostatin has beenrecognized as a means of neutralizing somatostatin in a target animaland thereby removing somatostatin's normal inhibitory effects on variousaspects of the animal's productivity, e.g., milk production in a dairycow. Reichlin S., ed., 1987, Somatostatin, Basic and Clinical Status,Plenum Press, New York (pp 3-50, 121-136, 146-156, 169-182, 221-228,267-274) Spencer G. S., 1985, Hormonal systems regulating growth,review, Livestock Production Science, 12, 31-46. Importantly, thesesomatostatin-based immunization procedures avoid the direct use ofanabolic hormones, e.g., growth hormone, and the like, in the animal andallow for small changes in the concentration of the endogenous anabolicfactors and thereby ecologically pure food products.

Somatostatin is known to have a relatively short half-life in the blood.In order to enhance the immunologic effects of somatostatin,immunization protocols have been developed to enhance the proteinshalf-life by conjugating somatostatin to target carrier proteins. Theseconjugated somatostatin proteins are designed to have increasedhalf-life and increased antigenicity in the blood and therefore provideenhanced benefits (especially in light of the cost of preparingsomatostatin). For example, chimeric somatostatin proteins are disclosedin U.S. Pat. No. 6,316,004, (and corresponding European PatentEP0645454) where various conjugated somatostatin-containing proteins areshown to have increased antigenicity and function with regard toproductivity of farm animals as compared to other conventionalimmunization or anabolic hormone-based procedures.

However, lower dose, higher antigenicity based immunization compositionsand procedures are needed to improve overall productivity and timelinessin the animal husbandry field. The present invention is directed towardproviding these more antigenic and functionally activesomatostatin-based immunization compounds, compositions and procedures.

Against this backdrop the following disclosure is provided.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptides, and thepolynucleotides that encode them, having enhanced immunogenicity ofsomatostatin. Polypeptides of the invention include somatostatin-14fused to a substantially inactivated chloramphenicol acetyl transferaseprotein via a functionally optimized linker. The chimeric polypeptidesof the invention provide highly effective and low cost materials for usein the animal husbandry field, as is described in more detail below.Embodiments of the invention include the amino acid and nucleic acidsequences as defined in SEQ ID NOs: 1-15.

The present invention also provides production and purificationprocedures for making the chimeric polypeptides of the invention in anendotoxin free and highly functional state. Endotoxin free polypeptidesprovide a substantial and unexpected advantage for use in certain targetanimals, where small amounts of endotoxin, typically thoughtadvantageous for eliciting an immunogenic response, actually lead tosignificant functional disadvantage. This is particularly the case whenpolypeptides of the invention are used to immunize United States bredand raised dairy cows. In addition, because the chimeric polypeptides ofthe invention show enhanced function as compared to conventionalmaterials, smaller and lower number of doses are used to immunize targetanimals. This decrease in required amounts also provides a resultantreduction of endotoxin in vaccines of the invention. The combination ofendotoxin free isolation and smaller use amount of the chimericpolypeptides of the invention allows for substantially endotoxin freevaccines to be used herein.

The invention also provides adjuvant compositions having enhancedfunction and safety as compared to conventional adjuvant materials.Adjuvants herein do not contain animal-derived materials and are free ofmost known chemical carcinogens, e.g., benzene and other like materials.Adjuvant compositions herein have proven to be unexpectedly effective ateliciting immune response when combined with target antigens.

The invention further provides vaccines containing the chimericpolypeptides of the invention with adjuvant compositions of theinvention. Vaccines herein are used for inducing immune responses invaccinated mammals and avian, for example, target farm animals.Illustrative farm animals for use herein include: dairy cows, pigs,sheep, goats, turkeys, rabbits, and bull calves. In some aspects thepolypeptides of the invention are prepared and purified with low or noassociated endotoxin. These vaccines have been optimized to elicit safeand enhanced immunogenic reactions in the target animal.

The invention further includes methods for vaccinating target mammalsand avians using the vaccines of the invention. Illustrative methods areprovided for vaccination of dairy cows to enhance milk production in asafe (for both animal and end-user), cost-effective and highly usefulmanner. Other illustrative methods include vaccination of piglets,sheep, turkeys, goats, rabbits or bull calves to enhance meat (andparticularly lean meat) production in a target animal in a safe andcost-effective manner.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description and areview of the appended claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustrative schematic of a pET30b CatSom plasmid inaccordance with embodiments of the present invention. The plasmidincludes a Kanamycin resistance marker, a Lac operator, T7 promoter, CATcoding sequence all in accordance with embodiments of the invention, alinker region in accordance with the invention herein and a somatostatinencoding region in accordance with the invention are also included.

FIG. 2 is an illustrative stained SDS-PAGE showing a 28KD bandcorresponding to the predicted size of a codon-optimized, CAT-defectivesomatostatin polypeptide of the invention. Lane 1 is LB+IPTG, reduced,Lane 2 is LB, reduced, Lane 3 is LB+IPTG and Lane 4 is LB.

FIGS. 3A and 3B are tables showing milk production in liters per day ofvaccinated (using vaccines as described in the Examples) and controldairy cattle (BSY). Each cow had a specific number identification asshown in each table.

IDENTIFICATION OF SEQUENCES AND SEQUENCE IDENTIFIERS

SEQ ID NO: 1 AGCKNFFWKTFTSC SEQ ID NO: 2GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT SEQ ID NO: 3 (His192 −> Gly,His193 −> Gly): Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag SEQ ID NO: 4: (His192 −> Gly, His193−> Gly): Mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvggavcdgfh vgrmlnelqq SEQ ID NO:5 (His193 −> Gly) Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag SEQ ID NO:6 (1 His193 −> Ala)Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatgctgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcag SEQ ID NO:7 (1 His + CAT wt)Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatggtgccgtttgtgatggcttccatgtcggcagaatgcttaatgaactgcagcag SEQ ID NO: 8 (one H −> G):Mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhgavcdgfh vgrmlnelqq SEQ ID NO:9 (H −> A) Mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhaavcdgfh vgrmlnelqq SEQ ID NO:10 tgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccgga attcatg SEQ IDNO:11 welhrsgprprprprpefm SEQ ID NO:12 welhrsgp(rp)nefm where n > 1 SEQID NO: 13 Atggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttggtggtgccgtttgtgatggcttccatgtcggccgtatgcttaatgaactgcagcagtgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatggccggctgcaagaacttcttttggaaaacctttacgagctgc SEQ ID NO:14mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvggavcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc SEQ ID NO: 15mekkitgyttvdisqwhrkehfeafqsvaqctynqtvqlditaflktvkknkhkfypafihilarlmnahpefrmamkdgelviwdsvhpcytvfheqtetfsslwseyhddfrqflhiysqdvacygenlayfpkgfienmffvsanpwvsftsfdlnvanmdnffapvftmgkyytqgdkvlmplaiqvhhavcdgfhvgrmlnelqqwelhrsgprprprprpefmagcknffwktftsc

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide chimeric polypeptides, andnucleic acid constructs that encode the same, having enhancedimmunogenicity of somatostatin. Embodiments herein include methods forpreparing the chimeric protein as well as methods for using the chimericprotein to increase certain productivity of target farm animals, andespecially for increasing milk production in dairy cows and meatproduction of cattle, pigs, sheep, rabbits, goats, bull calves, etc.

Embodiments of the present invention also include novel adjuvants foruse with the chimeric proteins of the invention for enhancedimmunogenicity in a target animal, enhanced safety for the target animalbeing vaccinated and enhanced safety for a user of the target animal,i.e., consumer of the vaccinated animal or consumer of a product fromthe vaccinated animal.

In one aspect, the invention provides chimeric somatostatin proteindesigned for enhanced immunogenicity of somatostatin. Chimericsomatostatin protein embodiments include an amino acid sequence ofsomatostatin-14 linked by a linker sequence to a substantiallyinactivated chloramphenicol acetyl transferase (CAT) truncated protein.In some cases the linker (also referred to herein as a spacer) has beenoptimized to enhance production of the chimeric polypeptide in targethost cells. In other cases, methods are provided for producing andisolating these enhanced amounts of chimeric protein in a substantiallyendotoxin free state.

In another aspect, the invention provides novel adjuvant compositions(which do not contain any animal derived proteins or chemicals, likebenzene) and vaccines that incorporate chimeric somatostatin proteins ofthe invention. Embodiments include unexpectedly effectiveadjuvant/chimeric somatostatin protein (typically substantiallyendotoxin free), i.e., novel vaccines, compositions for inducingantigenicity in vaccinated target farm animals. In some embodiments thetarget farm animal is a dairy cow, bull calve, pig, goat, etc. Note thatalthough the adjuvants of the present invention are described incombination with the chimeric somatostatin proteins herein, it isenvisioned that the inventive adjuvants herein could be used with othervaccines and in a variety of target animals, e.g., humans, pigs, dogs,cats, etc.

In other aspects, methods are provided for vaccinating target farmanimals using as few as one dose of this novel vaccine to obtainenhanced productivity, and in particular, enhanced milk production indairy cows and other like farm animals. These improved dose procedures(fewer number of vaccinations and smaller concentration of chimericsomatostatin) provide for time effectiveness and lower costs, which whenextended to the dairy industry represents a significant advancement infarm animal productivity. Also, the methods herein avoid use ofrecombinant growth hormone therapy which are a concern within the healthcare industry, i.e., both to the target farm animal, the end user andthe environment where excretion of recombinant growth hormone isreleased into the ground water supply.

DEFINITIONS

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

The term “amino acid” refers to any of the twenty naturally occurringamino acids as well as any modified amino acid sequences. Modificationsmay include natural processes such as posttranslational processing, ormay include chemical modifications which are known in the art.Modifications include but are not limited to: phosphorylation,ubiquitination, acetylation, amidation, glycosylation, covalentattachment of flavin, ADP-ribosylation, cross-linking, iodination,methylation, and the like.

The terms “chimeric polypeptide” or fusion protein refer to a firstpolypeptide having attached a second, heterologous polypeptide, suchthat the first and second polypeptides are expressed in frame. Often thetwo polypeptides can be attached via a linker or spacer segment tooptimize expression and function of the chimeric polypeptide(s) of theinvention.

The term “endotoxin” refers to toxins associated in the cell walls ofgram negative bacteria. In some cases the toxins are lipopolysaccharidecomponents of bacterial membranes.components of outer membrane of gramnegative bacteria cell walls.

The term “host cell” or “host cells” refers to cells established in exvivo culture. It is a characteristic of host cells discussed herein thatthey be capable of expressing the chimeric proteins of the invention.Examples of suitable host cells useful for aspects of the inventioninclude, but are not limited to, bacterial, yeast, insect and mammaliancells. Specific examples of such cells include SF9 insect cells (Summersand Smith, 1987, Texas Agriculture Experiment Station Bulletin, p 1555),E. Coli cells (BL21(DE3), Novagen), yeast (Pichia Pastoris, Invitrogen)and human liver cells (Hep G2 (ATCC HB8065).

The term “nucleic acid sequence” refers to the order of sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of the amino acidsalong a polypeptide chain. The deoxyribonucleotide sequence codes forthe amino acid sequence.

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers.

The term “substantially” refers to a “great extent,” for example,substantially removed means that at least 75%, more typically at least80%, 85%, 90%, 95% and most typically 96%, 97%, 98%, 99% of the targetmaterial is removed; substantially inactive means at least 75%, moretypically 80%, 85%, 90%, 95% and most typically 96% 97%, 98%, 99% of anenzyme is inactivated. As such a substantially inactive CAT enzyme isone that has 75%, 80%, 85%, 90%, 95% 96%, 97%, 98%, 99% or 100% of itsactivity removed. (CAT activity can be determined using known functionalassays, for example binding of n-Butyryl Coenzyme A to radiolabelledchloramphenicol and subsequent measurement by Liquid ScintillationCounting (LCS); by determining the amount of radioactive labeltransferred from [⁴Cacetyl CoA to chloramphenicol by thin layerchromatography (see Molecular Cloning: A Laboratory Model, 3^(rd) ed., JSambrook and D W Russell, 2001. Cold Spring Harbor Press] or other knownor like assays).

The term “vector” is used in reference to nucleic acid molecules thattransfer DNA segment(s) from one cell to another cell. The term“vehicle” is sometimes used interchangeably with vector. Two commontypes of vectors are plasmids and viral vectors.

Somatostatin

A number of studies have shown that animals immunized with somatostatinhave an average daily gain of 10-20%, an appetite reduced by 9% and an11% increase in the efficiency of food utilization. Animals immunizedwith somatostatin, and also their offspring, have correct proportions,and the distribution of the weight of the animals between the muscles,bones and fat is the same as in control animals (see Reichlin, 1987).Therefore, somatostatin immunization provides a useful and safe way ofenhancing a target animal's productivity. This is particularly the casewhen compared with use of recombinant growth hormone, which use hasraised concerns over hormone in milk or m eat from treated animals orfor the safely of the animals themselves or for build-up of the hormonein the ecosystem, particularly the ground water supply.

Somatostatin-14 is a biologically active tetradecapeptide produced inthe hypothalamus and gastrointestinal tract. The amino acid sequence ofthe tetradecapeptide is AGCKNFFWKTFTSC (SEQ ID NO: 1). The sequence ofsomatostatin-14 is highly conserved among mammals (Lin X W et al.Evolution of neuroendocrine peptide systems: gonadotropin-releasinghormone and somatostatin. Comp Biochem Physiol C Pharmacol ToxicolEndocrinol. 1998 119(3):375-88.) The tetradecapeptide is encoded by anucleic acid sequence GCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGT (SEQ IDNO: 2) (note that other nucleic acid sequences can be used to code SEQID NO: 1, however, SEQ ID NO: 2 is provided for illustrative purposes).

Somatostatin-14 is known to have a strong inhibitory effect on a largenumber of hormones involved in the growth and utilization of food inanimals. As previously described in U.S. Pat. No. 6,316,004(incorporated herein by reference for all uses), somatostatin-14 andchimeric versions of somatostatin can be used in immunization of animalsfor increase in daily weight and, where appropriate, milk production.These immunization procedures were preformed with conventional adjuvantsand did not utilize the somatostatin-based materials of this invention.Note that treatment of target animals with anti-somatostatin antibodieshas proven to be overly costly and functionally non-dramatic, therebyeliminating direct antibody treatment as non-practical. Muromtsev G. S.,et al., 1990, Basics of agricultural biotechnology, Agropromizdat,Moscow, pp 102-106. One aspect of the present invention is based on theconcept that the anti-somatostatin antibodies formed by compositions andmethods described herein attenuate but do not completely eliminate themostly inhibitory actions of somatostatin in the target animal. Thisprocess produces a natural and proportional increase in growth andproductivity in immunized target animals.

As such, aspects of the present invention facilitate somatostatin basedimmunization by providing highly immunogenic materials for use inimmunizing target animals. These somatostatin based immunizationcompounds have been optimized for expression and antigenicity. In someembodiments, somatostatin-14 is expressed as a codon-optimized,CAT-deficient somatostatin chimeric polypeptide. These materials providean unexpected improvement over other immunization based vaccines.

Novel Codon-Optimized, CAT-Deficient Somatostatin Constructs

One aspect of the invention provides isolated nucleic acid moleculesthat encode chimeric proteins having optimized somatostatin immunogenicactivity. In particular, embodiments of the invention include novelnucleic acid constructs that encode CAT fusion proteins havingimmunogenic activity for somatostatin. These polypeptides have beenidentified for optimal functional activity in immunization procedures.

In one embodiment, a construct having a schematic as shown in FIG. 1 isprovided to encode the chimeric polypeptides of the invention. Nucleicacid constructs of the invention generally encode an inactive CAT enzymewithout 10 C-terminal amino acids and includes one or two histidinereplaced amino acids. Substantial inactivation of CAT avoids adversereactions to CAT in the injected target animal (and thereby avoidance ofCAT resistance). Note that chloramphenicol is a commonly used antibioticin the cattle industry and resistance to chloramphenicol in cattle wouldbe adverse to the safety of animals vaccinated using materials of theinvention. As such, constructs having an inactivated CAT have theimmunogenicity of CAT without the adverse side-effects of resistance invaccinated animals (a problematic safety issue for the animals).

The CAT enzyme is inactivated by removing the imidazole group of His193(His195 in the canonical CAT_(III) variant, see Lewendon et al below).In another embodiment the CAT enzyme is inactivated by removing theimidazole groups of both His193 and nearby His192 (respectively His195and His194 for CAT_(III)). Removal of the essential His193 (His195 inCAT_(III)) imidazole group from the active site of CAT and replacementwith a alanine, glycine or other like amino acid results in substantialinactivation of the CAT enzyme (Lewendon A et al. (1994). Replacement ofcatalytic histidine-195 of chloramphenicol acetyl transferase: evidencefor a general base role for glutamate. Biochemistry. 33(7): 1944-50.).Finally, embodiments herein can also include CAT enzyme inactivationthrough removal of the imidiazole group of His192 alone (His194 forCAT_(III)). As for His193, replacement can be with an alanine, glycineor other like amino acid.

In some aspects, the one or more replaced histidine amino acids areencoded by nucleic acids located at position numbers 574-576 and 577-579of SEQ ID NO: 3 (corresponding to amino acid numbers 192 and 193 in SEQID NO:4). In some embodiments the nucleic acid sequences of theinvention include SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:7. Chimericproteins of the invention that include the histidine replaced constructsherein provide highly immunogenic proteins with little or no CATactivity, a significant improvement over the existing art. Thesubstantially inactivated CAT enzyme embodiments are attached to asomatostatin polypeptide of the invention. This attachment can be madedirectly or with a linker (as described more fully below).

Inactivation of the target sites (His192 and/or His193) can beaccomplished via any number of known procedures to those skilled in theart including site-directed mutagenesis and synthetic gene assembly. Inone embodiment, the nucleic acid sequence that encodes histidine 193 ismodified to encode an alanine, glycine or other like amino acid. Inanother embodiment, the nucleic acid sequences that encodes histidine192 is modified to encode alanine, glycine or other like amino acids.Typical combination replacements for the 192 and 193 chimericpolypeptide include: alanine, alanine; alanine, glycine; glycine,alanine; and glycine, glycine.

Embodiments of the present invention also include the amino acidsequences for CAT deficient polypeptides of the invention, includingamino acid sequence having SEQ ID NO: 8, 9 and 4 (corresponding tohis->gly at 193, his->ala at 193, and his->gly at both 192 and 193).

As shown in FIG. 1, the substantially non-active CAT enzyme can belinked to somatostatin-14 via a variable length spacer. The spacer beingrequired to insure presentation of the encoded somatostatin on a globalsurface. Spacer embodiments herein provide for optimal proteaseresistance and for optimal epitope exposure and inclusion in chimericpolypeptides of the invention have shown unexpected improvement overconstructs not having the linker sequence(s) of the present invention.

Spacer embodiments, therefore, have been optimized in length andcomposition to ensure CAT-defective somatostatin expression in variousmicroorganisms, and in particular in E. Coli. Original constructs asdescribed in U.S. Pat. No. 6,316,004, included a spacer having rare E.Coli codons and required the co-expression of rare tRNAs from a secondor helper plasmid. Spacer embodiments herein remove these rare E. Colicodons and thereby remove the need for a second helper plasmid, animprovement over previous technology.

In typical embodiments, the spacer has a nucleic acid sequence oftgggaactgcaccgttctggtccacgcccgcgccctcgcccacgtccggaattcatg (SEQ IDNO:10). One example of a spacer of the invention has an amino acidsequence of welhrsgprprprprpefm (SEQ ID NO: 11). A typical amino acidsequence for a spacer of the invention is welhrsgp(rp)_(n)efm where n>1(SEQ ID NO: 12). As noted above, these novel spaces sequences providefor enhanced protease resistance (thereby allowing for increasedproduction as compared to constructs disclosed in U.S. Pat. No.6,316,004) and optimal somatostatin-14 exposure. The combination ofsomatostatin-14 attached to a substantially inactivated CAT enzyme(histidine replaced constructs) by an optimally configured linker showedunexpected and surprising improvement over other conventional materialswhen used to immunize target animals for enhanced productivity.

Although not as optimal in function, other linker sequences of variablelength can be used to attach the somatostatin-14 to the substantiallyinactive CAT enzyme. In addition, it is envisioned that a direct fusionof the somatostatin-14 to the substantially inactive CAT enzyme couldalso be used herein, and is considered to be within the scope of thepresent invention.

Vectors and Host Cells

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally include aselectable marker and origin of replication, for the propagation host ofinterest. Host cells are genetically engineered to include these vectorsand thereby express the polypeptides of the invention. Generally,vectors herein include polynucleotides molecules of the inventionoperably linked to suitable transcriptional or translational regulatorysequences, such as those for microbial or viral host cells. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, mRNA ribosomal binding sites, and appropriate sequences whichcontrol transcription and translation. Nucleotide sequences are operablylinked when the regulatory sequences herein functionally relate to thechimeric polypeptide encoding polynucleotides of the invention.

Typical vehicles include plasmids, yeast shuttle vectors, baculovirus,inactivated adenovirus, and the like. In one embodiment the vehicle is amodified pET30b CatSom plasmid (see FIG. 1). Target host cells for useherein include bacterial host, e.g., E. Coli., yeast, SF-9 insect cells,mammalian cells, plant cells, and the like.

In one embodiment, the regulatory sequences include a T7lac, CAT, Trp,or T5 promoter for expression of the chimeric polypeptides of theinvention in E. coli or other like microbes. These regulatory sequencesare known in the art and are used under appropriate and knownconditions.

Where generically modified green plant cells are utilized forexpression, systems as developed by Planet Biotechnology and others canbe utilized.

Various plasmids of the invention have been constructed for expressionof chimeric polypeptides of the invention through utilization of targetregulatory sequences. Illustrative plasmids can include a T7lac promoter(see FIG. 1).

Host cells for expression of target chimeric polypeptides includeprokaryotes, yeast and higher eukaryotic cells. Illustrative prokaryotichosts include bacteria of the genera Escherichia, Bacillus, andSalmonella as well as the genera Pseudomonas and Streptomyces. Intypical embodiments the host cell is of the genera Escherichia and canbe Escherichia Coli (E. Coli).

As shown in the Examples below, constructs of the invention provide foroptimal CAT deficient somatostatin expression under a variety ofconditions. These constructs are particularly efficient for expressionin prokaryotic hosts and in particular bacteria of the generaEscherichia. Note as well that various plant expression systems can alsobe used in the context of the present invention, typically usingAgrobacterium trameficies.

Endotoxin Free Fusion Protein Purification

Aspects of the present invention include use of endotoxin free,codon-optimized, CAT-deficient somatostatin for use in vaccination ofanimals, and in particular for vaccination of farm animals, which insome cases are United States bred dairy cows. Endotoxin free materialsare particularly important for cattle bred and raised in the UnitedStates (see for example, Drackley, J K 2004. Physiological adaptationsin transition dairy cows. Pp 74-87 in Proc. Minnesota Dairy Herd HealthConf., St Paul, Minn. University of Minnesota, St. Paul).

In one embodiment, the chimeric immunogenic somatostatin-comprisingproteins of the invention are prepared by transforming target cells withappropriate somatostatin-containing vehicles. As noted above, vehiclesfor use herein include known plasmid and vector systems suitable forexpression in selected target cells.

In an aspect of the invention, chimeric immunogenicsomatostatin-comprising proteins are expressed in target host cells.Chimeric protein expression is performed using target regulatorysequences. In some aspects the chimeric polypeptides have been optimized(especially with regard to spacer sequences disclosed herein) forexpression in E. Coli.

Chimeric protein can then be purified in accordance with known proteinpurification technologies, including, for example, lysozyme lysis,differential centrifugation of inclusion bodies, sieve chromatographyand the like. Refolding procedures can be conducted in guanidinechloride and urea at alkaline pH followed by dialysis andlyophilization.

In one embodiment, E. coli cells are transformed using thecodon-optimized, CAT-deficient somatostatin containing plasmid—pET30bCatSom; the pET30b CatSom having appropriate E. Coli base regulatorysequences for expression. In some cases, fermentation of approximatelyten liters of these cells provides at least 500 grams and in some cases600 grams of total biomass, yielding about 4-6 grams of total protein.It is estimated from silver and coomassie blue staining that up to halfof the protein can be chimeric protein (see Example 2 and FIG. 2).

In some embodiments herein, chimeric protein of the invention ispurified from transformed host cells in a substantially endotoxin freestate. Realization that endotoxin, and in particular multiple exposuresto endotoxin, in some animals, and in particular dairy cows, results insubstantially compromised animals (mastitis and endotoxin shock in dairycows bred and raised in the United States) was an unexpected andsurprising result that the present inventors obtained. This realizationresulted in an attempt to remove or lower the endotoxin dose amount ornumber of exposures in dairy cow vaccinations. Note that this endotoxinbased effect is much less realized in cows bred and raised in Russia andother countries as the dairy cattle are descendent from a differentstrain of cow. Holstein Association, 1 Holstein Place, Brattleboro, Vt.05302-0808. This finding in United States dairy cows is generallycontrary to the expectation that a vaccine should include some lowamount of endotoxin to help maximize an animals' immune response, as isthe case for dairy cows when vaccinated with somatostatin in some otherEuropean markets (see U.S. Pat. No. 6,316,004).

As such, some embodiments herein are directed at production ofsubstantially endotoxin free chimeric proteins for use in vaccines, andespecially for use in vaccines used in the cattle industry and used inthe cattle industry within the United States. In certain embodiments theendotoxin levels are at or below 1 EU/ml and in other embodiments theendotoxin levels are substantially eliminated, i.e., the chimericpolypeptides of the invention are substantially endotoxin free.

In one embodiment, recovered IB from lysed host cells is washed multipletimes using a wash solution devoid of endotoxin, i.e., endotoxin freewater or solution. The recovered IP pellet can optionally be washeduntil endotoxin levels are below approximately 1 EU/ml (endotoxin testscan be performed using one or more known assays, including commerciallyavailable test kits from MP Biochemicals, Charles River, etc.). In someembodiments the wash solution is endotoxin free and includes one or moreproteolytic protein inhibitor(s), e.g., phenylmethanesulphonylfluoride(PMSF), 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF), etc. In someembodiments the wash solution is phosphate buffered saline (PBS) havingan inhibitory effective amount of PMSF, AEBSF or a combination of bothPMSF and AEBSF.

In some aspects, substantially endotoxin free pellets can be treatedwith a protein unfolding solution at pH 12.5 containing urea andrefolded in a protein refolding solution containing a reduced molarityof urea with arginine, glycerol and/or sucrose. Purified chimericprotein concentration is modified to be between 1 and 3 mg/ml andtypically about 1.4 to 1.8 mg/ml. In some cases, substantially endotoxinfree chimeric protein is provided to vaccine formulations at about 1.5to 5 mg/2 ml dose and more typically from 2.0 to 3.5 mg/2 ml dose.

Other endotoxin removal procedures are envisioned to be within the scopeof the present invention and can include, for example, commerciallyavailable ion-exchange endotoxin removal columns, hydrophobic columns,etc (see for example Mustang E or G Columns (Millipore)).

Enhanced Immune Response Adjuvant

Embodiments of the invention provide new adjuvants for enhancedinduction of humoral immunity. These adjuvants provide a significantimprovement over conventional materials for the induction of a humoralresponse. Adjuvants herein can be used with numerous vaccines, but areshown in the Examples in use with polypeptides of the invention forvaccination in dairy cows, pigs or bull calves.

Importantly, all components of adjuvants herein are of non-animalorigin, thereby eliminating potential cross-contamination of vaccinatedanimals from potentially contaminated adjuvant components. For example,embodiments herein can utilize animal origin free Tween 80. This isparticularly important when the target animal is a dairy cow, due toconcerns over bovine spongiform encephalopathy (BSE) or other likebovine ailments. Note that these concerns are equally appropriate forhuman treatment where non-animal origin adjuvant provide significantsafety benefits. Additionally, adjuvant embodiments herein are free ofbenzene and other like carcinogenic compounds. These embodiments providea safety benefit not available in most conventional adjuvant compounds.For example, embodiments herein can utilize Carbopol® 974P or benzenefree polycyclic acid.

In one embodiment, the immunological adjuvant comprises an oil-in-wateremulsion in combination with selected antigens admixed within anemulsion premix.

Illustrative oil-in-water emulsions for use herein include combinationsof mineral oil, Tween 80, Span 85 and target polymers (benzene-freePolyacrylic acid). In some cases the target polymer is selected from thegroup consisting of Carbomer Homopolymer Type B. Typical oil-wateremulsions comprise from about 8-10% mineral oil (v/v), 0.003 to 0.004%Tween 80 (v/v), 0.007 to 0.008 Span 85 (v/v) and 0.04 to 0.06% polymer(w/v).

Illustrative emulsion premixes of the invention are composed of a highmolecular weight polymer, surfactant, and emulsifier in at approximate50% oil-aqueous base. High molecular weight polymers for use hereininclude acrylic acids crosslinked with allyl ethers of pentaerythritol.In some cases the high molecular weight polymers have a Brookfield RVTviscosity of between about 29,000 and 40,000, for example, Carbopol®974P (Noveon, Inc).

Method for Obtaining Optimized Immune Response in Dairy Cows or OtherTarget Farm Animals

In accordance with compositions and methods of the present invention,the immunogenic compositions described herein (endotoxin free,codon-optimized, CAT-deficient somatostatin constructs) are combinedwith novel adjuvants, as described above, to provide vaccines of theinvention. In one embodiment, endotoxin free, codon-optimized, CATdeficient somatostatin constructs at a total dose of 2.98 mg/2 ml (ofwhich from 5% to 25% is adjuvant (v/v), more typically from 10% to 20%is adjuvant and most typically about 20% is adjuvant) is administered.Note that other conventional adjuvants are envisioned to be within thescope of the invention and can be used with the codon-optimized,CAT-deficient somatostatin constructs of the invention, however, optimalresults have been shown when the novel adjuvants herein are used in thiscapacity.

The purpose of the novel somatostatin constructs and adjuvant is toincrease the productivity of a target animal, typically a farm animal,and more typically a dairy cow, bull calve, sheep, pig, or goat.

The preparation is injected intramuscularly or subcutaneously,preferably fewer than 12 times, more preferably fewer than 6 times, andin some cases as few as only one time. When more than one injection isrequired in a target animal, an interval of 14 to 28 days is typicalbefore the next injection. As noted above, embodiments herein avoid theuse of recombinant hormone treatment to target animals, a major benefitin the animal husbandry field (recombinant growth hormone has beenassociated with early onset of puberty in girls and variousenvironmental concerns that the manure from treated cattle can adverselyeffect both surface and groundwater environments).

Sterile compositions of the invention can be administered bysubcutaneous or intramuscular routes. In typical cases the site ofadministration is the target animal's neck or tail, although other sitesmay be utilized. Note that a site should be used such that an adversereaction does not impede the animal's ability to move, eat, drink, etc.

As noted above, vaccines herein, typically in an endotoxin free state,using the novel adjuvants described herein, provide a significantimprovement in meat and milk production of dairy cows, cattle, pigs,etc. These treatments, however, are not accompanied by an increase infeed consumption.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Construction of CAT-Defective Somatostatin Fusion Protein

The present example illustrates the production of a CAT-defectivesomatostatin fusion protein in accordance with embodiments of thepresent invention. Site-directed mutagenesis was performed on plasmidpET30b-Cat-Som to replace His192 and His193 with glycine residues (aftermodification: Gly192 and Gly193). Inactivation of the His193 (andHis192) residues eliminates the capacity of the CAT enzyme to acceptprotons, thereby providing complete inactivation of the CAT.

The spacer in the same pET30b-Cat-Som (having the His replacement(s))was codon-optimized for expression by E. coli in the absence ofco-expressed tRNA molecules.

The modified CAT-defective somatostatin nucleic acid construct is shownas SEQ ID NO: 13. The CAT-defective somatostatin fusion protein sequenceis disclosed as SEQ ID NO: 14, being compared to an unmodifiedCAT-somatostatin fusion protein (SEQ ID NO: 15).

Example 2 CAT-defective Somatostatin Fusion Protein can be Expressed atHigh Levels

The codon-optimized CAT-defective somatostatin construct as described inExample 1 was used to express the fusion protein in BL21(DE3) cells.Transformed cells were grown in LB and induced with 0.4 mM IPTG forapproximately three hours. One milliliter of cells from a density of OD0.7 culture were pelleted, and heated at 70° C. for ten minutes in 100μl SDS sample buffer. A sample of 40 μl of cell extract was loaded perlane for SDS PAGE.

As shown in FIG. 2, a 28 KD band corresponding to the predicted size ofa codon-optimized, CAT-defective somatostatin fusion protein was visiblein lanes 1 (LB+IPTG, reduced) and 3 (LB+IPTG) after induction with IPTG.No expression is seen in control lanes 2 (LB, reduced) and 4 (LB). Asexpected, there was no difference in fusion protein size when run understandard or reducing conditions.

Example 3 Endotoxin Free, Codon-Optimized CAT-Deficient SomatostatinContaining Vaccine

An illustrative vaccine in accordance with the present invention:

a. JH 14 (Adjuvant)   24 ml mineral oil - 50% (v/v) Tween 80 - 0.1694%(v/v) Span 85 - 0.1915% (v/v) Carbopol 974NP - 0.125% (w/v) b. RefoldedProtein of the invention 95.6 ml (2.96 mg/ml) - see Example 2 c.Phosphate Buffered Saline 35.6 ml d. Antibacterial/Antifungal 0.36 mo 120 ml

Example 4 Endotoxin Free Chimeric Peptides/Adjuvants Provide IncreasedMilk Production

A random pool of dairy cows (Holstein Crosses—US bred and raised) wasidentified, each was 31 to 65 days post-calving (3^(rd) through 5^(th)lactation). Each cow was examined and determined to be in optimal healthby a veterinarian.

The average cow weight in the study was from about 1,000 to 1,200 lbs.Six lactating cows were treated with 1.96 mg/chimeric protein/2 ml dosein JH14. Alternatively, 9 lactating cows were provided with aconventional rBST treatment. Treatments and milk production study wasconducted at a large scale, intense milk production dairy.

Vaccinations were conducted at day 0. Anti-SST serum antibodies andIGF-1 serum levels tested at 4 weeks, Milk production and identificationof general health of animals were conducted on a regular schedule.

Six cows that were vaccinated using inventive compositions describedherein had a normal appearance, with no endotoxin reaction or foodwithdrawal. All six cows had a positive serologic response to SST with amean titer of 1:14. Milk production of the six cows was obtained withonly one vaccination (see FIG. 3A), showing a mean yield increase of23.7%.

Nine cows treated using conventional rBST injections at 0 and 14 dayswith an overall mean increase in milk productivity of 2% (see FIG. 3B).

The data in this Example shows the drastic improvement in effectivenessfor using the endotoxin free constructs in combination with inventiveadjuvants in dairy cows. These results are dramatically improved towardthe animal's health and productiveness as compared to cows rejected twotimes with rBST.

Example 5 Endotoxin Free Chimeric Peptides/Adjuvants Provide IncreasedMeat Production In Piglets of Treated Sows

A random pool of sows will be identified, each being at least 35-36 daysprior to farrowing. Each sow will be examined and determined to be inoptimal health by a veterinarian. Pregnant sows will be immunized twotimes using vaccines of the invention (see Example 3), once at 35-36days prior to delivery, and once at 8 days prior to delivery. A controlgroup of pregnant sows will be maintained for comparison purposes (novaccinations or vaccination with sterile saline).

Delivered piglets from the vaccinated group will have greatersurvivability and be of a greater average size. It is the increase inpiglet size that enhances the percent survivability, as larger pigletsare less likely to be pushed away from the sow's teat. Treated andcontrol piglets will be weighed at day 21, day 30 and day 75.Vaccinations of the present invention will increase the piglet dailyweight by an average of 35% over the course of the 75 day period.

Importantly, piglet survivability and weight are increased through useof the vaccines of the present invention in the absence of recombinantgrowth hormone. This is a significant improvement over recombinanthormone therapy.

Example 6 Endotoxin Free Chimeric Peptide/Adjuvants Provide IncreasedMeat Production in Treated Bull Calves

A random pool of bull calves, one to three months of age, will beidentified, and injected with compositions of the invention. Weightincrease over a period of approximately ten months will be monitored andcompared to a control group, the control group being treated the same inevery sense as the injected group except for the vaccine injections ofthe invention. Each bull calve will be examined and determined to be inoptimal health by a veterinarian over the course of the treatments.

Injections herein for the vaccinated group are performed at zero weeks,4 weeks and 8 (three total vaccinations). Vaccinations will be providedsubcutaneously or intramuscularly to the neck using 18-21 gauge ccneedle. Booster injections were also provided (4 boosts, three boosts orno boosts). Vaccination injections included 2 mg/2 ml of the chimericpolypeptide. The chimeric polypeptide was prepared as described hereinhaving both histidine residues in CAT replaced with glycine amino acidsof the invention and a optimized linker as described by SEQ ID NO: 4.

Vaccinated bull calves and control calves are each weighed to take aninitial weight. It is expected that vaccinated animals herein will showa 15 to 40% weight increase over control animals. This increase inaverage weight for treated bull calves shows a significant advantageover no treatment.

Importantly, harvested meat from treated bull calves does not containrecombinant growth hormone.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

1. A chimeric polypeptide having the immunogenicity of somatostatin,comprising: an amino acid sequence of somatostatin-14 linked to asubstantially inactive and truncated chloramphenicol acetyl transferasepolypeptide wherein the somatostatin- 14 is linked to the inactivechloramphenicol acetyl transferase by a spacer, the spacer optimized foroptimal expression of the chimeric polypeptide in a target host cell andwherein the inactive chloramphenicol has at least histidine residue 193replaced with an alanine, glycine or other like amino acid.
 2. Thechimeric polypeptide of claim 1 wherein the spacer has an amino acidsequence of SEQ ID NO: 11 or SEQ ID NO:
 12. 3. The chimeric polypeptideof claim 1 wherein the substantially inactive and truncatedchloramphenicol acetyl transferase polypeptide has an amino acidsequence of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO
 9. 4. The chimericpolypeptide of claim 1 having an amino acid sequence with at least 99%sequence identity with the amino acid sequence of SEQ ID NO:
 13. 5. Thechimeric polypeptide of claim 1 having an amino acid sequence of SEQ IDNO:
 13. 6. An immunogenic composition comprising the chimericpolypeptide of claim 1 together with a pharmaceutically suitableadjuvant in an amount effective to elicit an immune response.
 7. Animmunological adjuvant comprising an oil-in-water emulsion admixed withan emulsion premix wherein the oil-in-water emulsion comprises mineraloil, Tween 80, Span 85 and one or more polymers.
 8. The adjuvant ofclaim 7 wherein the emulsion premix comprises a high molecular weightpolymer, a surfactant, and an emulsifier in an oil-aqueous base.
 9. Theadjuvant of claim 7 further comprising pre-selected antigens wherein theantigens are native, recombinant or synthetic polypeptides,polysaccharides and glycoproteins.
 10. An immunogenic compositioncomprising the chimeric polypeptide of claim 1 together with theadjuvant if claim
 8. 11. A method for increasing milk production indairy cows comprising: vaccinating a dairy cow with one or more doses ofthe composition of claim 6; and allowing for at least ten days duringwhich a dairy cow's milk production will increase as compared to thesame cow's milk production in the absence of the vaccination.
 12. Themethod of claim 11 wherein the dairy cow is vaccinated with only onedose of the composition of claim
 6. 13. A method for increasing milkproduction in dairy cows comprising: vaccinating a dairy cow with one ormore doses of the composition of claim 10; and allowing for at least tendays during which a dairy cow's milk production will increase ascompared to the same cow's milk production in the absence of thevaccination.
 14. A method for increasing lean meat production in a farmanimal comprising; vaccinating the farm animal with one or more doses ofthe composition of claim 10; and allowing for several weeks during whichthe farm animal lean meat production will increase relative to lean meatproduction in a similar farm animal in the absence of the vaccination.